The present invention employs an inducible bacterial repressor proteins to block transcription of an exogenously-added gene in mammalian cells. The addition of exogenous inducer substances to the cells induces the repressor thus removing the block to transcription and derepressing expression of the exogenously-added gene. Both the lexA and lac repressor have been used in mammalian cells to control gene expression. See for example, Brown et al., Cell 49:603-612 (1987); Hu et al., Cell, 48:555-566 (1987); and Smith et al., EMBO J., 7:3975-3982 (1988).
Chimeric gene constructions have been prepared in which the lac repressor is used to inhibit expression of the simian virus 40 (SV40) early promoter, a vaccinia virus promoter, or a T3 bacteriophage in mammalian cells where the promoter contains appropriately placed operators (the repressor recognization sequences). In these systems, repression is removed by the addition of a lac inducer, typically isopropyl-.beta.-D-thiogalactoside (IPTG). Brown et al., supra: Figge et al., Cell, 52:713-722 (1988); Fuerst et al., Proc. Natl. Acad. Sci. U.S.A., 86:2549-2553 (1989); and Hu et al., supra. However, induction by the addition of IPTG did not produce necessary increases in the level of gene expression of the promoter-controlled eukaryotic gene when present in the form of a stably integrated gene in a eukaryotic cell.
An alternative expression system involved preparation of a chimeric protein having the specificity of a bacterial repressor and the induction capacity of a mammalian transactivator protein. The first reported chimeric protein comprised the lexA DNA-binding protein and the activation domain of the yeast transcription factor, GAL4. Brent et al., Cell, 43:719-736 (1985). More recently a chimeric regulatory protein was produced containing the E. coli lac repressor that was modified to include a nuclear localization signal from the simian 40 (SV40) large tumor antigen and was fused with the transcription activation domain from the herpes simplex virus type 1 virion protein 16. This chimeric protein, called lac activator protein (LAP), was a potent activator of promoters containing lac operator sequences positioned either upstream or downstream from the transcription unit. See Lapow et al., Mol. Cell. Biol., 10:3343-3356 (1990). The uninduced LAP protein is not a repressor but rather acts as an activator of gene expression until induced by an inducer. The LAP protein, although expressed in a target cell's cytoplasm and transported to the nucleus where it activates a target gene, is not an expression system in which gene transcription is enhanced through the use of endogenously added inducers or other externally imposed regulatory switch mechanisms.